Osteoporosis is one of the leading causes of mortality and disability in older women. Osteoporosis causes 68,000 deaths per year, which is equivalent to the number of deaths from breast cancer and all gynecological cancers combined. One of the most effective strategies to prevent osteoporosis is to replace estrogen at the onset of menopause. Unfortunately, prolonged treatment with estrogen leads to adverse effects, including an increased incidence of breast and endometrial cancer. Clearly, estrogens that retain their beneficial effects on bone, but do not elicit a proliferative effect on breast or endometrial cells, have the potential to become first-line drugs to prevent osteoporosis. One approach to discover safer and more selective estrogens is to dissect the molecular mechanisms whereby estrogens activate and repress gene transcription. In contrast to transcriptional activation, very little is known about the mechanisms of repression. We studied how estrogens and phytoestrogens repress the tumor necrosis factor (TNF-alpha) promoter, because TNF-a causes osteoporosis by stimulating osteoclasts to resorb bone. Based on these studies, we hypothesized that estrogen receptor (ERbeta) is more effective than ERalpha at repressing gene transcription, and that the activation function-2 surface of ERs and coactivator proteins are required for estradiol-mediated repression. Whereas these findings are novel and important, their interpretation is limited, because they were done with the TNF-alpha promoter linked to reporter genes in transient transfected cells. The goals of this proposal are to extend these observations to the native TNF-alpha gene, and to further probe the basic molecular mechanisms whereby ERs repress gene transcription. In this proposal we will use chromatin immunoprecipitation assays to characterize the protein-protein interactions that occur at the native TNF-alpha promoter between estrogen receptors, transcription factors, such as c-jun and NFKB, and p160 and p300 coregulatory proteins in bone cells that are stably transfected with ERalpha or ERbeta controlled by a tetracycline-inducible promoter. We hypothesize that identifying the proteins involved in repression, and determining how these factors interact with each other is key to developing repression-selective estrogens. We believe that repression-selective estrogens have the potential to become first-line drugs for preventing osteoporosis, because we hypothesize that these estrogens will prevent osteoporosis, but will not promote breast or endometrial cancer.